Method for detecting invasive microvescles derived from tumor cells

ABSTRACT

The present application relates to the isolation and analysis of populations of microvesicles, such as populations of microvesicles that are shed by tumor cells and contain the protein ARF6. Invasive microvesicles from tumor cells contain a variety of specific proteins, including ARF6.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Grant No.5R56CA115316-02 awarded by the National Cancer Institute. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present application relates generally to microvesicles and theirisolation and analysis, including methods of identifying populations ofinvasive microvesicles that contain the protein ARF6. As described inthe examples below, invasive microvesicles from tumor cells contain avariety of specific proteins, including ARF6.

2. Description of the Related Art

Microvesicles are small, membrane-enclosed structures that are shed froma variety of cell types and can contain a variety of bioactivemolecules, including nucleic acids and proteins. Microvesicle sheddingby the outward fission of membrane vesicles from the cell surface is aselective process that occurs more frequently in certain cells, such ascancerous tumor cells. These released microvesicles (also referred to asmicroparticles, particles, and ectosomes) have been widely detected invarious biological fluids, including peripheral blood, urine, saliva,and ascites.

Microvesicles are believed to facilitate various processes, includingtumor invasion and metastasis, and are also believed to play a role ininflammation, coagulation, stem-cell renewal and expansion, evasion ofthe immune response, and bone mineralization. The composition ofmicrovesicles, and thus their function, varies depending on the cellsfrom which they originate. For example, microvesicles secreted byskeletal cells have been found to play a role in initiating bonemineralization, while microvesicles secreted from endothelial cells havebeen implicated in angiogenesis. Microvesicles are thought to play arole in metastasis by facilitating angiogenesis, escape from immunesurveillance, and extra-cellular matrix (ECM) degradation. Proteolyticactivity associated with microvesicles shed by tumor cells has beenfound to correlate with disease stage.

The process of metastasis occurs when cells detach from a primary tumorand invade surrounding tissues to reach other, distal locations. Thisprocess leads to the formation of secondary tumors, and is one of thelife-threatening hallmarks of malignant cancer. Standardized screeningmethods and techniques that are sensitive enough to detect early-stagecancer and other diseases are currently unavailable for a variety ofconditions, including ovarian, prostate, breast, glioma, and melanomacancers. As a result, metastasis often occurs before a patient can bediagnosed and treated.

Recent studies show that a variety of molecules are involved with thecomplex process of metastasis. One such molecule is the protein known asARF6, of the ARF family of small GTP-binding proteins, which regulatesmembrane trafficking and actin cytoskeleton remodeling and has a role inacquisition of migratory and invasive potential of cancer and other celltypes. Recent studies utilizing in vitro cell invasion assays haveindicated that in invasive melanoma, glioma, and breast cancer celllines, the ARF6 GTP/GDP cycle can regulate the invasive potential of thecells. In addition, cellular depletion of ARF6 by siRNA or inhibition ofARF6 activation by expression of a dominant negative ARF6 mutantattenuates tumor cell invasion in vitro. Recent animal studies have alsorevealed a role for ARF6 activation in melanoma and glioma cell invasion(V. Muralidharan-Chari et al., Cancer Res. 69, 2201-09 (2009), B. Hu etal., Cancer Res. 69, 794-801 (2009)). Moreover, screening of variousbreast tumor cell lines reveals a direct correlation between ARF6protein expression and invasive capacity (S. Hashimoto et al., Proc.Natl. Acad. Sci. 101, 6647-52 (2004)). In addition, a molecule known asARF6 exchange factor GEP 100 is expressed in 70% of primary breastductal carcinomas, and is preferentially co-expressed with EGFR inmalignant tumors (M. Morishige et al., Nat. Cell Biol. 10, 85-92(2008)).

SUMMARY OF THE INVENTION

The present application relates to the isolation, identification andanalysis of populations of microvesicles, such as invasive microvesiclesthat are shed by tumor cells. As described in the examples below,invasive microvesicles from tumor cells contain a variety of specificproteins, including ARF6. Thus, in some embodiments the isolatedmicrovesicles comprise the protein ARF6.

In some embodiments, invasive microvesicles are identified in a sampleby centrifuging the sample to collect a population of microvesicles andassaying the prepared population of microvesicles for the protein ARF6,wherein detecting the protein ARF6 identifies the population ofmicrovesicles as invasive microvesicles. Identification of invasivemicrovesicles can be used, for example, to assess the invasiveness of atumor in a patient.

In other embodiments, invasive microvesicles are identified in a sampleby preparing a population of microvesicles from the sample and assayingthe prepared population of microvesicles for the presence of one or moreproteins selected from the group consisting of ARF6, Vamp3, MHC class I,MT1-MMP, β1-integrin and β1-integrin receptor.

In some embodiments, the sample is a biological fluid. In someembodiments, the biological fluid is blood, serum, plasma, urine,saliva, or ascites. In some embodiments, the biological fluid isobtained by washing an anatomical structure of a subject, and thencollecting the wash fluid after it has come in contact with theanatomical structure.

In some embodiments, the microvesicles are lysed prior to analysis. Inother embodiments, the microvesicles are not lysed, and are analyzedwhole. In some embodiments, the microvesicles are analyzed by contactingthem with an antibody that binds to a protein being detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows vesicles released into the growth media from 2.2×10⁶ LOX,LOXARF6-GTP, and LOXARF6-GDP cells, isolated and probed for ARF6 and β1integrin by western blotting. FIG. 1B shows the same results calculatedby protein quantification. The average of three separate experiments isshown with standard error bars. Cell lysates were probed for ARF6 andα-tubulin by western blotting. Lower and higher molecular weight ARF6bands in LOXARF6 cells correspond to endogenous and HA-tagged exogenousARF6, respectively.

FIG. 1C shows LOXARF6-GTP and LOXARF6-GDP cell lines analyzed byelectron microscopy. The image in the top panel shows heterogeneousvesicular structures near the surface of LOXARF6-GTP cells. Lower panelshows vesicular structures that appear to stud the surface ofLOXARF6-GDP cells. Bar: 1000 nm.

FIG. 1D shows vesicles released into the growth media from an equivalentnumber (1.5×10⁶ cells) of indicated tumor cell lines in culture thatwere isolated and probed for ARF6 by western blotting.

FIG. 2A shows microvesicles shed by parental LOX and LOXARF6-GTP celllines that were fractionated as described. Equivalent amounts of proteinfrom each fraction were probed for ARF6 content. Endogenous ARF6 andHA-tagged mutant ARF6 are enriched only in the 10,000 g fraction.

FIG. 2B shows fractionated vesicle populations isolated from LOXARF6-GTPcells analyzed by whole mount electron microscopy. Microvesiclesisolated in the 10,000 g fraction are larger (300-900 nm) andheterogeneous relative to the more uniform 50-70 nm vesicles in the100,000 g fraction, typical of exosomes. Bar: 500 nm.

FIG. 3A shows microvesicles shed from an equal number of parental LOXand LOXARF6-GTP cells. The cells were isolated, and microvesicle lysateswere analyzed by gelatin zymography. Cell lysates were probed forα-tubulin expression. High and low molecular weight gelatinases arepresent in microvesicle lysates.

FIG. 3B shows lysates of LOXARF6-GTP cells or of shed microvesicles thatwere analyzed for indicated endogenous proteins or transfected proteins(EGFP-VAMP3 or EGFP-VAMP7) by western blotting.

FIG. 4A shows parental LOX and LOXARF6-GTP cell lines that were treatedwith 30 μM U0126 and microvesicles shed into the media that wereisolated and quantitated for protein. Data from 5 independentexperiments is plotted. Standard error bars are shown.

FIG. 4B shows parental LOX, LOXARF6-GDP, and LOXARF6-GTP cells that weresubjected to subcellular fractionation. The amount of endogenousphospho-ERK and total-ERK in the membrane and cytosolic fractions wasanalyzed by immunoblotting.

FIG. 5A shows lysates of LOX cells that were transiently transfectedwith HA-tagged-ARF6-N48I mutant protein. The cells were probed forphospho-ERK and total ERK by western blotting. The data isrepresentative of 3 separate experiments. Band intensities werequantified by densitometry, and the ratio of phospho-ERK to total ERK isshown.

FIG. 5B shows microvesicles released into the media by parental LOX andLOX cells transfected with ARF6-N48I. Microvesicles were isolated bydifferential centrifugation and quantitated for protein content. Theaverage of four separate experiments with standard error bars is shown.Microvesicles in the growth media of cells expressing ARF6-N48I is lowerrelative to non-transfected controls.

FIG. 5C shows LOX cells that were treated with 0.3% t-butanol or1-butanol for 30 min, and then fixed and stained for actin to visualizesurface-associated microvesicles. For each experimental condition, cellsthat showed eight or more microvesicles at the surface were scored, andthe percent inhibition of vesicle shedding was determined. 250 cellswere observed for each experiment, and the data from four independentexperiments is plotted. Standard error bars are shown.

FIG. 5D shows lysates of LOXARF6-GDP cells treated with propranolol (0.1mM) and atenolol (10 uM). The treated cells were probed for phospho-ERKand total-ERK. Band intensities were quantified by densitometry. Theaverage of 3 independent experiments is plotted and standard error barsare shown.

FIG. 5E shows LOXARF6-GDP cells treated with propranolol or atenolol.The treated cells were visualized, and those with microvesicles presentat the cell surface were scored as described above.Propranolol-treatment of LOXARF6-GDP cells restored vesicle shedding.

FIG. 6A shows parental LOX, LOXARF6-GTP, and LOXARF6-GDP cells inculture that were incubated with fresh media containing 2 μM LatrunculinA (Lat A) for 45 min. Released microvesicles were isolated andquantitated for protein. The average of three independent experimentswith standard error bars for each experimental condition is shown.

FIG. 6B shows cell lysates of LOX, LOXARF6-GTP, and LOXARF6-GDP celllines that were analyzed for endogenous phospho-MLC by western blotting.α-tubulin expression was also assessed as an indicator for equalloading. Band density was measured by densitometric scanning.

FIG. 6C shows lysates of LOX cells treated with U0126 (30 μM) that wereanalyzed for endogenous phospho-MLC, phospho-ERK, total-ERK, andα-tubulin by western blotting. Additional lanes on the immunoblotbetween lanes 2 and 3 were spliced out. Band density was measured bydensitometric scanning.

FIG. 6D shows LOXARF6-GDP cells that were treated with the followinginhibitors: Go6976 (PKC inhibitor-10 μM), ML-7 (MLCK inhibitor-10 μM),U0126 (MEK inhibitor-30 μM) and SB203580 (p38 inhibitor-10 μM) for 2 hrsat 37° C. Cell lysates were probed for phospho-MLC, phospho-ERK andα-tubulin by western blotting.

FIG. 7A depicts a working model for ARF6-mediated regulation of cellinvasion. In this model, activated ARF6 facilitates tumor cell invasionby promoting invadopodia formation and microvesicle shedding. Theformation of invadopodia and microvesicle release requires ARF6 and ERK.

FIG. 7B depicts the signaling pathway downstream of ARF6-regulated ERKactivation that is required for microvesicle shedding. ARF6-regulatedERK localization to the plasma membrane, as well as its activation atthe plasma membrane, both require PLD. ERK-induced phosphorylation ofMLCK, in turn, promotes MLC phosphorylation, which is required foractomyosin-based membrane fission, leading to microvesicle release.

FIG. 8 shows the average fluorescence intensity of cortactin ininvadopodia and microvesicles. LOX cells were plated on FITC conjugatedgelatin and allowed to invade overnight. These cells were fixed andstained with anti-cortactin antibody and rhodamine phalloidin tovisualize invadopodia and microvesicle structures. The fluorescenceintensity of the cortactin signal was measured in invadopodia (n=56) andmicrovesicle structures (n=62). The signal intensity was normalized tothe signal noise in an area of the slide free of cells. Error barsrepresent ±1 standard deviation.

FIG. 9A depicts ARF6-mediated regulation of vesicle shedding in SW480colon carcinoma cells. Vesicles released into the growth media from2.2×10⁶ SW480, SW480ARF6-GDP, and SW480ARF6-GTP cells were isolated bylow speed centrifugation and probed for ARF6 by western blotting.Corresponding cell lysates were probed for ARF6 and α-tubulin. Low andhigher molecular weight ARF6 bands correspond to endogenous andHA-tagged exogenous protein, respectively.

FIG. 9B also depicts ARF6-mediated regulation of vesicle shedding inSW480 colon carcinoma cells. SW480 cells with and without ARF6 (T27N)expression, as noted, were analyzed by electron microscopy. Imagesreveal the presence of heterogeneous microvesicle-like structures at thetumor cell surface. ARF6 (T27N) expression appears to block themicrovesicle release. Bar: 1000 nm.

FIG. 10A shows that MLC phosphorylation is required for microvesicleshedding in parental LOX and LOXARF6-GTP cells. Parental LOX andLOXARF6-GTP were treated with 10 μM ML-7 for 2 hrs. Microvesiclesreleased were isolated, quantitated for protein, and the data is shownas fold change in shedding relative to untreated LOX cells. MLCphosphorylation is blocked by the MLCK inhibitor in LOX and LOXARF6-GTPcells but not in LOXARF6-GDP cells.

FIG. 10B shows MLCK-dependent phosphorylation of MLC in parental LOX andLOXARF6-GTP cells, but not in LOXARF6-GDP cells. Cell lines as indicatedwere treated with ML-7, and cell lysates were probed for phospho-MLC andα-tubulin by Western blotting. Band density was measured bydensitometric scanning. Fold increase in the ratio of phospho-MLC toα-tubulin in ML-7 treated cells relative to untreated cells is shown.

FIG. 10C shows that MLC phosphorylation is independent of PKC inparental LOX and LOXARF6-GTP cells. Lysates of parental LOX andLOXARF6-GTP treated with Go6976 (10M) for 2 hrs at 37° C. were probedfor phospho-MLC and α-tubulin by Western blotting.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

ARF6 has been suggested to play a role in tumor cell invasion andmetastasis. ARF6 may promote cell invasion in a variety of ways,including through regulation of protease secretion. As discussed herein,via its effects on phospholipid metabolism and ERK activation, ARF6regulates protease release by modulating the shedding of plasmamembrane-derived microvesicles into the surrounding environment.Structural and biochemical characterization of ARF6-positivemicrovesicles shed by tumor cells shows that the protease cargocontained within the microvesicles is functionally robust and promotesextracellular matrix degradation. ARF6 also maneuvers the actomyosinmachinery of cells to facilitate shedding of microvesicles from thetumor cell surface into the environment surrounding the cells. Thesefindings are significant, particularly in light of reports demonstratingthat proteolytic activities of microvesicles shed by tumor cellscorrelate directly with malignancy and invasiveness in cancer and otherdiseases (V. Dolo et al., Clin. Exp. Metastasis 17, 131-40 (1999), L. E.Graves et al., Cancer Res. 64, 7045-49 (2004)).

As described in the examples below, invasive microvesicles from tumorcells contain one or more specific proteins. In some embodiments,invasive microvesicles comprise ARF6. Other proteins present in invasivemicrovesicles may include one or more of Vamp3, MHC class I, MT1-MMP,β1-integrin and β1-integrin receptor. These proteins can be used toidentify invasive microvesicles in a biological sample, such as a sampleof biological fluid taken from a patient suffering from or suspected ofsuffering from a tumortdentification of a population of invasivemicrovesicles in a sample from a patient can be used, for example, toidentify the presence of an invasive tumor in the patient and to assessthe invasiveness of a known tumor.

Collection and Preparation of Microvesicles

A sample from a subject can be analyzed for the presence ofmicrovesicles and, in particular, for the presence of invasivemicrovesicles. In some embodiments, the subject is a mammal. In otherembodiments, the subject is a human. In some embodiments, the sample isa biological fluid from the subject. In some embodiments, the sample maybe taken from a patient suffering from or suspected of suffering from atumor. In other embodiments, the sample may be taken from a subjectbelieved to be healthy. In some embodiments, a biological sample istaken from a subject suffering from a tumor, and the sample is takenfrom a region close to or associated with the tumor. In otherembodiments, a biological sample is taken from a region located awayfrom or unrelated to the tumor.

As microvesicles have been previously identified in blood, serum,plasma, urine, saliva and ascites, in some embodiments samples of one ormore of these fluids are taken from a patient. Samples can be obtainedusing any of a variety of standard techniques. For example, blood orascites can be removed from a patient with a needle and syringe, or anysuitable suction device. In some embodiments the biological sample isnot a fluid produced by the subject, but rather a fluid brought intocontact with the subject. For example, a sample may be obtained bywashing an anatomical structure and subsequently collecting the fluidthat passed over the structure. This technique is often used duringsurgical procedures to collect samples of cells that may be present invarious regions of a patient's body, such as the abdominal cavity.Further, a sample may be obtained by swabbing an anatomical structure,such as the inside of a patient's cheek. A sample may be subject tofurther processing, such as separating plasma from whole blood, prior toanalyzing the sample for the presence of microvesicles and in particularfor the presence of invasive microvesicles.

Once a sample is obtained it is centrifuged to separate themicrovesicles, if present, from other components. The speed, duration,and temperature of centrifugation can be varied in order to obtain thedesired results. In some embodiments, a sample is washed and centrifugedat about 5,000 g to 15,000 g at least once, preferably two or moretimes. In some embodiments the sample is centrifuged for about 10 toabout 60 minutes, for example for about 10 minutes, 15 minutes, 20minutes, or 25 minutes. In other embodiments, a sample is centrifugedfor 35 minutes, 40 minutes, or 45 minutes. In a particular embodiment, asample is centrifuged at about 10,000 g for about 30 minutes. In someembodiments, the sample is not centrifuged at a speed higher than about30,000 g.

In other embodiments, a sample is first centrifuged at a lower speed,for example about 2,500 g prior to the higher speed centrifugation atabout 5,000 g to 15,000 g as described above. In some embodiments, asample is first centrifuged at about 1,200 g to about 3,000 g prior tothe higher speed centrifugation. In some embodiments this firstcentrifugation removes cell nuclei, debris, and larger particulatematter from the sample. In some embodiments, a sample is firstcentrifuged at about 2,500 g for about 5 to about 30 minutes, followedby centrifugation at about 10,000 g as described above. Thus, in oneembodiment the sample is centrifuged at about 2,500 g for 15 minutes,followed by centrifugation at 10,000 g for 30 minutes.

In some embodiments, the centrifugation steps are typically conducted ata temperature of less than about 10° C., such as about 2-10° C. However,in some other embodiments, the centrifugation steps are conducted at atemperature of about 8-18° C.

After centrifugation, the isolated microvesicles can be prepared foranalysis. In some embodiments, the microvesicles are washed in phosphatebuffered saline or another suitable solution. After washing, themicrovesicles can be lysed using any of a variety of appropriate lysisbuffers, such as RIPA buffer, that are well known to those of skill inthe art. In some embodiments, the microvesicles are not lysed, but areanalyzed whole.

Analysis of Microvesicle Contents

After collection of the microvesicles as described above, any of avariety of experimental techniques can be used to analyze them for thepresence of one or more proteins selected from the group consisting ofARF6, Vamp3, MHC class I, MT1-MMP, β1-integrin and β1-integrin receptor.The presence of the assayed proteins indicates that the sample comprisesinvasive microvesicles.

In some embodiments, the microvesicles are assayed to determine if ARF6is present. The presence of ARF6 and, typically, one or more additionalproteins identifies the microvesicle population as comprising invasivemicrovesicles.

In some embodiments, microvesicles are assayed for the presence of ARF6and one or more additional proteins selected from Vamp3, MHC class I,MT1-MMP, β1-integrin and β1-integrin receptor. In some embodimentsinvasive microvesicle populations are identified based on the presenceof ARF6 and MT1-MMP.

In other embodiments, the presence of VAMP3, either alone or incombination with one or more additional proteins selected from ARF6, MHCclass I, MT1-MMP, β1-integrin and β1-integrin receptor, is used toidentify a population of microvesicles as comprising invasivemicrovesicles.

The microvesicles can be assayed for the presence of other combinationsof proteins selected from selected from ARF6, Vamp3, WIC class I,MT1-MMP, β1-integrin and β1-integrin receptor. For example, they can beassayed for the presence of ARF6 and any combination of one or more ofVamp3, WIC class I, MT1-MMP, β1-integrin and β1-integrin receptor. Someexemplary combinations directed to ARF6 and one or more additionalproteins are described below. Similar combinations can be assayed forsubstituting Vamp3 for ARF6. In all cases, the presence of thecombination of proteins is indicative of a population of invasivemicrovesicles.

In some embodiments, the microvesicles are assayed for ARF6 as well asVamp3 protein. In other embodiments, the microvesicles are assayed forARF6 as well as WIC class I protein. In some embodiments, themicrovesicles are assayed for ARF6 as well as β1 integrin. In otherembodiments, the microvesicles are assayed for ARF6 as well as β1integrin receptor. In other embodiments, the microvesicles are assayedfor ARF6 as well as MT1-MMP.

In some embodiments, the microvesicles are assayed for ARF6, Vamp3, andWIC class I protein. In other embodiments, the microvesicles are assayedfor ARF6, Vamp3, and β1 integrin. In other embodiments, themicrovesicles are assayed for ARF6, Vamp3, and β1 integrin receptor. Inother embodiments, the microvesicles are assayed for ARF6, Vamp3, β1integrin and MT1-MMP. In other embodiments, the microvesicles areassayed for ARF6, Vamp3, β1 integrin receptor and MT1-MMP. In otherembodiments, the microvesicles are assayed for ARF6, Vamp3, and MT1-MMP.In other embodiments, the microvesicles are assayed for ARF6, Vamp3, WICclass 1 and MT1-MMP. In other embodiments, the microvesicles are assayedfor ARF6, Vamp3, WIC class I, and β1 integrin. In other embodiments, themicrovesicles are assayed for ARF6, Vamp3, WIC class I, and β1 integrinreceptor. In other embodiments, the microvesicles are assayed for ARF6,Vamp3, β1 integrin, and β1 integrin receptor. In other embodiments, themicrovesicles are assayed for ARF6, Vamp3, WIC class I, β1 integrin andMT1-MMP. In other embodiments, the microvesicles are assayed for ARF6,Vamp3, WIC class I, β1 integrin receptor and MT1-MMP. In otherembodiments, the microvesicles are assayed for ARF6, Vamp3, β1 integrin,β1 integrin receptor and MT1-MMP.

In other embodiments, the microvesicles are assayed for ARF6, MHC classI, β1 integrin, and β1 integrin receptor. In other embodiments, themicrovesicles are assayed for ARF6, MHC class I, β1 integrin, β1integrin receptor and MT1-MMP. In some embodiments, the microvesiclesare assayed for ARF6, MHC class I, and β1 integrin. In some embodiments,the microvesicles are assayed for ARF6, MHC class I, and β1 integrinreceptor. In some embodiments, the microvesicles are assayed for ARF6,MHC class I, β1 integrin and MT1-MMP. In some embodiments, themicrovesicles are assayed for ARF6, MHC class I, β1 integrin receptorand MT1-MMP. In some embodiments, the microvesicles are assayed forARF6, MHC class I, and MT1-MMP. In some embodiments, the microvesiclesare assayed for ARF6, β1 integrin, and β1 integrin receptor. In someembodiments, the microvesicles are assayed for ARF6, β1 integrin, β1integrin receptor and MT1-MMP. In some embodiments, the microvesiclesare assayed for ARF6, Vamp3, MHC class I, β1 integrin, and β1 integrinreceptor. In some embodiments, the microvesicles are assayed for ARF6,Vamp3, MHC class I, β1 integrin, β1 integrin receptor and MT1-MMP. Insome embodiments, the microvesicles are assayed for ARF6, β1 integrin,and MT1-MMP. In some embodiments, the microvesicles are assayed forARF6, β1 integrin receptor and MT1-MMP.

In some embodiments, the microvesicles are assayed for the presence ofone or more of the proteins of interest, such as ARF6, Vamp3, MHC classI, MT1-MMP, β1-integrin and β1-integrin receptor, by contacting themwith an antibody that specifically binds to the target protein that isbeing assayed. Antibodies to the target proteins are known in the artand are available from various sources. For example, anti-neutralizingβ1-integrin, AIIB2, is available from the Developmental StudiesHybridoma Bank at the University of Iowa, anti-MHC-class I is availablefrom Serotec, anti-MT1-MMP is available from Dr. M. C. Rio, France,anti-ARF6 is available from Dr. C. D'Souza-Schorey, Notre Dame, andanti-Vamp3 is available from Dr. P. Chavrier (Institute Curie, France).

In some embodiments, the microvesicles are assayed for the absence ofsome proteins. For example, in some embodiments the microvesicles areassayed for the absence of one or more of Vamp7, Rab8A, TfnR(transferrin receptor), cortactin, and Tks5. The absence of one or moreof these proteins indicates that the population of microvesicles isinvasive. In some embodiments, the absence of one or more of theseproteins, combined with the presence of one or more of ARF6, Vamp3, MHCclass I, MT1-MMP, β1-integrin and β1-integrin receptor indicates thatthe population of microvesicles is invasive.

In some embodiments, western blotting is used to identify the presenceor absence of target proteins in the microvesicle population, asdescribed below. Microvesicles can be prepared for western blottingusing standard methods.

In other embodiments, immunofluorescent staining and microscopy can beused to identify the presence of target proteins using standardprocedures. These methods can also be used to visualize the location anddistribution of various molecules in the isolated microvesicles. Forexample, whole (un-lysed) microvesicles can be plated onpoly-L-lysine-coated or gelatin-coated glass coverslips, and can then befixed and processed as previously described in V. Muralidharan-Chari etal., Cancer Res. 69, 2201 -09 (2009).

The total protein content of lysed microvesicles can be calculated, forexample by using the Bradford assay (BioRad). This assay utilizes aspectroscopic analysis procedure to measure the concentration of proteinin a solution.

Gelatinase activity of microvesicle contents can be determined, forexample using gelatin zymography. In this technique, microvesiclecontents are separated on SDS-PAGE gels containing gelatin, andgelatinase activity is measured as previously described in, for example,D. H. Manicourt et al., Anal. Biochem. 215, 171-79 (1993).

In addition to the gelatin zymography technique, an in vitro degradationassay can also be used to assess the ability of microvesicle contents todegrade gelatin. The gelatin degradation assay has been previouslydescribed in H. Hoover et al., Methods Enzymol. 404, 134-47 (2005).Alternatively, isolated microvesicles can be seeded and incubated ongelatin-coated coverslips for 6-8 hours to assess their gelatindegradation potential.

Electron Microscopy (EM) can also be used to visually assess cells andmicrovesicles. Conventional and whole mount electron microscopy can beperformed as described in, for example, G. Raposo et al., J. Exp. Med.183, 1161-72 (1996). Briefly, cells grown on coverslips are fixed with2.5% glutaraldehyde in 0.1M cacodylate buffer overnight and processed asdescribed.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES Example 1 Phenotypic Variations of LOXARF6-GTP and LOXARF6-GDPCell Lines

To investigate the mechanisms by which ARF6 promotes cell invasion, twodifferent experimental cell lines were used, each containing a differentARF6 mutation. One cell line, called LOXARF6-GTP, stably expresses anHA-tagged, GTPase deficient ARF6 mutant referred to as ARF6-Q67L. Theother cell line, called LOXARF6-GDP, stably expresses a dominantnegative ARF6 mutant referred to as ARF6-T27N. In addition to these twoexperimental cell lines, a parental LOX cell line that expressesnon-mutant ARF6 was also used. The most striking phenotype observed uponmicroscopic examination of LOXARF6-GDP cells was the presence ofvesicle-like bulbous structures that decorate the cell surface. Thesestructures were also seen on the surfaces of parental LOX andLOXARF6-GTP cell lines, although they were less readily obvious.Instead, in the latter two cell lines, vesicles were released into thegrowth medium. The lack of vesicles in the growth media of LOXARF6-GDPcells suggests that ARF6 activation is likely required for theirrelease. When collected by low speed centrifugation of the growth mediaand examined for total protein or probed for ARF6 expression, vesiclesshed from LOXARF6-GTP cells exhibit significantly more protein and ARF6,indicative of increased shedding from these cells relative to theparental cell line (FIG. 1A, FIG. 1B). Furthermore, mutant ARF6-GDP wasnot present on shed vesicles.

LOXARF6-GTP cells were seeded on gelatin-coated coverslips, and shedmicrovesicles were subsequently detected in the gelatin matrix at thecell surface. Furthermore, these vesicular structures appear to bedistinct from cortactin-positive invadopodia that extend into thegelatin matrix. Microvesicles do not contain cortactin, although they docontain β1 integrin, which is also present in invadopodia (E. T. Bowdenet al., Exp. Cell Res. 312, 1240-53 (2006)). Quantitation of cortactinin microvesicles relative to cortactin in invadopodia is shown in FIG.8. Some cells exhibit expansive membrane arbors decorated withmicrovesicles. Cells had a tendency to adopt this arborizationphenotype, suggestive of horizontal movement when the underlying gelatinis relatively thick (≧5 μm). It is possible that cells form invadopodiaat the adherent face initially and when they invade into substantialmatrix and move laterally, this arbor phenotype takes effect. Incontrast, LOXARF6-GDP cells appear bulbous when plated on gelatin,largely due to vesicles studded at the cell surface and little to nomatrix degradation underneath the cells. As reported previously,invadopodia protrusions at the adherent surface were not observed uponexpression of the dominant negative ARF6 mutant (S. E. Tague et al.,Proc. Natl. Acad. Sci. 101, 9671-76 (2004)). Thus, ARF6 activation iscoupled to two apparently distinct cellular processes linked to matrixinvasion; invadopodia formation, and the release of surface vesiclesinto the surrounding environment.

Morphological examination of LOXARF6-GTP and LOXARF6-GDP cell linesusing electron microscopy (EM) suggests that vesicles at the surface ofcells are heterogeneous in size (300-900 nm) (FIG. 1C). EM-basedinvestigations also indicate that cells display no signs of nuclearfragmentation or apoptosis. All of the above suggests that the ARF6GTPase cycle regulates the release of a heterogeneous population ofvesicles from tumor cells into the surrounding environment.

Vesicle shedding was examined in other tumor cell lines; SW480, a coloncarcinoma cell line, PC3, a prostate adenocarcinoma cell line, andMDA-MB-231, an invasive breast tumor cell line. Gross morphologicalexamination by phase contrast microscopy showed that shed vesicles werepresent in the growth media of all aforementioned cell lines. Analysisof shed vesicles collected from the growth media revealed the presenceof endogenous ARF6 on isolated vesicles (FIG. 1D). Furthermore, dominantinhibition of ARF6 function by expression of ARF6-T27N in these tumorcell lines prevented vesicle release (data with SW480 cell line is shownin FIG. 9A, FIG. 9B).

Example 2

ARF6-GTP Facilitates the Release of Plasma Membrane-DerivedMicrovesicles into the Surrounding Environment

There is accruing evidence for the existence of unconventional secretorymechanisms, such as the release of exosomes and microvesicles, that donot utilize the classical signal-peptide secretory transport pathway.Exosomes are internal vesicles of multivesicular bodies/late endosomesthat are released upon exocytosis (B. Fevrier et al., Curr. Opin. CellBiol. 16, 415-21 (2004)). Morphological characterization ofARF6-regulated shed vesicles suggests that they are microvesicles.Isolated ARF6-positive vesicles pellet by centrifugation atapproximately 10,000 g, unlike exosomes, which sediment bycentrifugation at approximately 100,000 g. FIG. 2A shows thatARF6-positive vesicles are not present in the 100,000 g fraction.Ultrastructural analyses of whole mount preparations of the 10,000 g and100,000 g fractions confirmed the heterogeneity of the 10,000 gmicrovesicles relative to the more uniform 50-70 nm vesiclescharacteristic of exosomes in the 100,000 g fraction (FIG. 2B).

Studies have shown that phosphatidylserine (PS) externalizationaccompanies shedding of plasma membrane-derived microvesicles (B. Hugelet al., Physiology (Bethesda) 20, 22-27 (2005)). Externalization of PSon the surface of microvesicles based on reactivity with Annexin-V, ahigh affinity PS-binding protein, has been found in some studies. Thiswas particularly evident in LOXARF6-GDP cells, where microvesicles studthe cell surface. These microvesicles are not apoptotic bodies. Asstated above, expression of mutant ARF6 in LOX cells does not inducemorphological changes such as condensed chromatin and pyknotic nuclei.In addition, data from expression of mutant ARF6 in LOX cells does notindicate that cleaved caspase 3 is present in the cells. On the otherhand, when treated with okadaic acid, a known inducer for apoptosis,experimental data indicates that cleaved caspase 3 is present in ahigher proportion of the cells. Accordingly, these data suggest thatARF6 activation facilitates the release of microvesicles.

Example 3 Shed Microvesicles Contain Proteases and Facilitate ECMDegradation

Previous studies have shown that microvesicles shed by breast carcinomaand ovarian cancer cell lines contain proteases, including matrixmetalloproteinases, such as MMP-2 and MMP-9 (V. Dolo et al., Cancer Res.58, 4468-74 (1998)). ARF6-positive microvesicles obtained from a 10,000g fraction collection procedure contained high and low molecular weightgelatinases, as indicated by gelatin zymography (FIG. 3A), and MT1-MMP.Notably, MT1-MMP silencing significantly decreases the basalinvasiveness of experimental cell lines. Moreover, shed microvesicles,when seeded on FITC-gelatin, were capable of matrix degradation, whichappeared as dark spots around microvesicle membranes.

Some researchers believe that β1 integrins facilitate interaction ofshed microvesicles with the ECM, and that vesicle contents are releasedafter microvesicle bursting induced by acidic pH of the tumorenvironment (G. Taraboletti et al., Neoplasia 8, 96-103 (2006)). Theaddition of inactivating β1 integrin antibody, AIIB2, to isolatedmicrovesicles or experimental cell lines blocked adhesion and matrixdegradation, suggesting that protein topology in the microvesiclemembrane is still maintained and that integrin receptor association withextracellular matrix is important for microvesicle-mediated matrixdegradation AIIB2 binds to and therefore labels the extracellularsurface of both cells and shed microvesicles, indicating that β1integrin is a component of the microvesicle membrane. Thus, whileproteases at the invadopodia surface facilitate pericellular proteolysisin the immediate vicinity of the cell, shed microvesicles containingproteases may travel away from the immediate cellular vicinity andfacilitate proteolysis at distal locations. Microvesicle release couldtherefore provide a mechanism for rapid and directed proteolysis thatcreates a path of diminished resistance for cell migration.

Example 4

Selective Sorting of Cargo into Cell Surface Microvesicles

Besides proteases, microvesicles have been shown to be selectivelyenriched in β1 integrin receptors and MHC class I molecules (MHC-I).Both MHC-I molecules and integrin receptors traffic to and from theplasma membrane via ARF6-regulated endosomal compartments (J. G.Donaldson, J. Biol. Chem. 278, 41573-76 (2003)). Microscopic examinationshows that MHC-I in LOXARF6-GDP cells is present in the perinuclearcompartments and the cell surface, but largely at the cell surface inLOXARF6-GTP cells. Western blotting analysis of shed vesicles revealsthat, along with endogenous ARF6, MHC-I, β1 integrin, VAMP 3, andMT1-MMP were also present in microvesicles (FIG. 3B). Of note, processedforms of MT1-MMP are present in the microvesicles. However,microvesicles were devoid of transferrin receptors, VAMP7, or Rab8A, aswell as cortactin and TksS, which are known components of invadopodia.Thus, cargo trafficked via recycling of endosomes to the cell surfaceappears to be selectively sorted into these surface microvesicles.

Example 5 ARF6-GTP-Induced Microvesicle Shedding Involves theExtracellular Signal Regulated Kinase (ERK)

ARF6-enhanced melanoma cell invasion is dependent on the activation ofthe extracellular signal-regulated kinase (ERK) (S. E. Tague et al.,Proc. Natl. Acad. Sci. 101, 9671-76 (2004)). Moreover the ARF6 GTPasecycle regulates ERK activation (S. E. Tague et al., Proc. Natl. Acad.Sci. 101, 9671-76 (2004), S. E. Robertson et al., Mol. Biol. Cell 17,645-57 (2006), J. S. Tushir et al., Embo. J. 26, 1806-19 (2007))downstream of c-Raf/A-Raf (E. Nekhoroshkova et al., PLoS ONE 4(2): e4647(2009)). Therefore, ARF6-GTP-induced microvesicle shedding wasinvestigated to determine if it is also dependent on ERK signaling.Treatment of parental LOX and LOXARF6-GTP cells with U0126, an inhibitorof MEK, the kinase immediately upstream of ERK, resulted in significantinhibition of basal as well as ARF6-GTP enhanced microvesicle shedding(FIG. 4A). Upon MEK inhibition, both parental LOX and LOXARF6-GTPaccumulated microvesicles at the cell surface that resembled theLOXARF6-GDP phenotype. Similarly, MEK inhibition also blockedmicrovesicle shedding from PC3 and SW480 cell lines. Thus,ARF6-regulated microvesicle shedding requires ERK.

Consistent with earlier findings in HeLa cells (S. E. Robertson et al.,Mol. Biol. Cell. 17, 645-57 (2006)), ERK localizes to the cytoplasm inLOXARF6-GDP cells. In contrast, membrane-associated ERK is predominantlyat the cell surface in LOX and LOXARF6-GTP cells. This becomes furtherevident in sub-cellular fractionation studies, wherein the activatedphospho-ERK distribution is significantly greater in the membranefraction of LOXARF6-GTP cells (FIG. 4B). Thus, ARF6 activation likelyenhances ERK redistribution to the plasma membrane, which in turnfacilitates its phosphorylation and microvesicle release.

Example 6 ARF6-Regulated ERK Activation Involves Phospholipase D

Studies have shown that ARF6 stimulates phospholipase D (PLD) activity(C. D'Souza-Schorey et al., Nat. Rev. Mol. Cell Biol. 7, 347-58 (2006)).Further, PLD has been shown to facilitate ERK activation. Here, ARF6regulated PLD activity and its involvement with ERK activation wastested. First, phospho-ERK levels upon PLD inactivation were examined.The results show that expression of ARF6-N481, an ARF6 mutant defectivein PLD activation, inhibits ERK activation (FIG. 5A) and microvesicleshedding (FIG. 5B). PA levels were significantly decreased inARF6-N48I-expressing cells, and ERK distribution was found inintracellular cytoplasmic structures. Next, primary alcohols were usedto inhibit PLD, which results in the formation of phosphatidylalcohol atthe expense of phosphatidic acid by replacing water. The results showthat treatment of cells with 1-butanol also inhibits ERK activation andmicrovesicle shedding (FIG. 5C). Thus, PLD activation is upstream of ERKactivation and is therefore involved with ARF6-regulated microvesicleshedding.

Next, the role of phospho-ERK levels in microvesicle shedding wasinvestigated. Specifically, microvesicle shedding was examined inLOXARF6-GDP cells after phospho-ERK levels were restored, or afterstimulating PLD activity through pathways independent of ARF6. Treatmentwith propranolol, a PA phosphohydrolase inhibitor (O. A. Jovanovic etal., Mol. Biol. Cell 17, 327-35 (2006)), restored phospho-ERK levels inLOXARF6-GDP cells, as shown in FIG. 5D and induced microvesicle sheddingin LOXARF6-GDP cells (FIG. 5E). Unlike propranolol, another GCPRantagonist, atenolol, did not have any effect on phospho-ERK levels ormicrovesicle release (FIG. 5D, FIG. 5F). These data corroborate theinvolvement of PLD and ERK activation in regulating microvesicleshedding. Finally, the effect of 1-butanol on microvesicle sheddinginduced by expression of constitutively activated MEK was investigated.Activated MEK increases microvesicle shedding more than 12-fold comparedto the parental LOX cells; however, 1-butanol had little to no effect onmicrovesicle shedding, confirming that ERK lies downstream of PLDactivation.

Example 7 ERK Facilitates Microvesicle Shedding by PhosphorylatingMyosin Light Chain

Treatment of LOX cells with blebbistatin, a small molecule inhibitor ofmyosin II activity, or with latrunculin A, an actin-binding toxin thatinhibits actin polymerization, profoundly affected microvesicleshedding. In the presence of blebbistatin, microvesicles do not form.85-90% of latrunculin A treated cells exhibited clustered microvesiclesat the cell surface. Accordingly, latrunculin A treatment also inhibitedthe release of microvesicles from both parental LOX and LOXARF6-GTPcells (FIG. 6A). Thus, microvesicle shedding is likely mediated via anactomyosin-dependent mechanism. MLCK, a Ca²⁺/calmodulin-dependentkinase, phosphorylates myosin II light chain (MLC) to promotecontraction of the actin-based cytoskeleton (A. Sobieszek et al.,Biochem. J. 295 (Pt 2), 405-11 (1993)). ERK has been shown tophosphorylate MLCK, which in turn phosphorylates MLC at Thr18/Ser19 andthereby stimulates MLC activity (R. L. Klemke et al., J. Cell Biol. 137,481-92 (1997), D. H. Nguyen et al., J. Cell Biol. 146, 149-64 (1999)).ARF6-GTP-induced ERK activation at sites of vesicle release was tested,and the results indicate that it leads to localized activation of MLCK,which in turn stimulates serine phosphorylation of MLC to allow forcegeneration required for microvesicle fission. In support of thiscontention, immunofluorescent labeling of LOX tumor cells revealed thatphospho-MLC localizes to the “necks” of microvesicles at the cellsurface.

Phosphorylation of MLC was examined in parental LOX and LOX mutant linesby western blotting. As shown in FIG. 6B, levels of phospho-MLC aresignificantly increased in LOXARF6-GTP cells compared to parental LOXcells. Treatment of parental LOX and LOXARF6-GTP cells with ML-7, anMLCK inhibitor, blocks MLC phosphorylation as well as microvesicleshedding (FIG. 10A, FIG. 10B, FIG. 10C). Taken together, these dataindicate that ARF6 facilitates microvesicle fission via a mechanism thatinvolves ERK-dependent MLC phosphorylation.

As seen in FIG. 6B, no decrease in phospho-MLC levels was observed inLOXARF6-GDP cells. In fact, phospho-MLC levels in LOXARF6-GDP cells wereslightly higher than basal levels. A possible explanation for thisobservation is that the phosphorylation of MLC in LOXARF6-GDP cells is“inhibitory” and independent of ERK. To investigate this further, thelevels of phospho-MLC in the presence and absence of U0126 were comparedin parental LOX and LOX mutant lines. As seen in FIG. 6C, inhibition ofphospho-MLC was evident in the presence of MEK inhibitor in parental LOXand in LOXARF6-GTP cells, but there was no inhibition in phospho-MLClevels in LOXARF6-GDP cells. Also, treatment with ML7 had little to noeffect on MLC phosphorylation in LOXARF6-GDP cells (FIG. 10A, FIG. 10B,FIG. 10C). This further confirms that the phosphorylation of MLC inLOXARF6-GDP cells is not mediated by ERK.

Alternative pathways were also examined, such as the activation of PKCor p38 (P. L. Goldberg et al., Am. J. Physiol. Lung Cell Mol. Physiol.282, L146-54 (2002), N. V. Bogatcheva et al., Am. J. Physiol. Lung CellMol. Physiol. 285, L415-26 (2003)), for MLC phosphorylation inLOXARF6-GDP cells. To distinguish between PKC and p38-mediatedphosphorylation of MLC, LOXARF6-GDP cells were treated with SB203580, ap38 inhibitor, or Go6976, a PKC inhibitor. Results showed thatphospho-MLC levels were decreased specifically upon PKC inhibition (FIG.6D). Thus, MLC phosphorylation in LOXARF6-GDP occurs via PKC, and isindependent of phospho-ERK. Results also indicated that MLCphosphorylation is independent of PKC in parental LOX and in LOXARF6-GTPcells, as no decrease in phospho-MLC levels was observed in the presenceof PKC inhibitor. Thus, in LOXARF6-GDP cells, MLC is phosphorylated byPKC, which is inhibitory, while in LOXARF6-GTP cells, MLCphosphorylation is regulated upstream by ERK, a step that promotesMLC-regulated contraction, and in turn, microvesicle fission (FIG. 7A,FIG. 7B). In sum, ARF6-GTP facilitates microvesicle fission via amechanism that involves ERK-dependent MLC phosphorylation, and ARF6-GDPhas the opposite effect via PKC-induced phosphorylation of MLC.

Example 9 Detection of Invasive Microvesicles in a Biological Sample

In this example, a sample is obtained from a patient suspected of orknown to have a tumor. A needle is inserted into the area of thesuspected or known tumor and a sample of the ascitic fluid is collectedin a syringe. A portion of the fluid is then centrifuged first at about800 g for about 10 minutes, then at about 2,500 g for about 15 minutes,then at about 10,000 g for 30 minutes. During the centrifugationprocess, the sample is maintained at a temperature of about 2-8° C.Following the centrifugation process, isolated microvesicles are washedin phosphate buffered saline. Western blot analysis is used to determinethe presence or absence of one or more of ARF6, Vamp3, MHC class I,MT1-MMP, β1-integrin and β1-integrin receptor in the microvesicles. Thepresence of ARF6 protein, and, optionally, one or more of Vamp3, MHCclass I, MT1-MMP, β1-integrin and β1-integrin receptor in the isolatedmicrovesicles identifies the population of microvesicles as comprisinginvasive microvesicles.

1. A method of identifying invasive microvesicles in a biologicalsample, the method comprising the steps of: providing a biologicalsample; centrifuging the biological sample to collect a population ofmicrovesicles, and assaying the population of microvesicles for presenceof the protein ARF6, wherein the presence of ARF6 identifies thepopulation of microvesicles as comprising invasive microvesicles.
 2. Themethod of claim 1, wherein the biological sample is a biological fluidobtained from a subject.
 3. The method of claim 2, wherein thebiological fluid comprises one or more of blood, plasma, serum, urine,saliva, or ascites.
 4. The method of claim 2, wherein the biologicalfluid is obtained by washing an anatomical structure of the subject andcollecting the wash fluid after it has come in contact with theanatomical structure.
 5. The method of claim 1, wherein assaying theprepared population of microvesicles is accomplished by contacting themicrovesicles with an antibody that binds to ARF6.
 6. The method ofclaim 1, additionally comprising the step of: assaying the preparedpopulation of microvesicles for the presence of one or more proteinsselected from the group consisting of Vamp3, WIC class I, β1-integrinand β1-integrin receptor.
 7. The method of claim 7, wherein thepopulation of microvesicles is assayed by western blot.
 8. The method ofclaim 1, wherein the sample is centrifuged at about 10,000 g.
 9. Themethod of claim 8, wherein the sample is centrifuged at about 2,500 gprior to centrifugation at about 10,000 g.
 10. A method of analyzing apopulation of microvesicles for the presence of invasive microvesiclescomprising: determining whether the protein ARF6 and the protein MT1-MMPare present in the population of microvesicles, wherein the presence ofARF6 and MT1-MMP indicates that invasive microvesicles are present inthe sample.
 11. The method of claim 10, additionally comprisingdetermining whether one or more additional proteins selected from thegroup consisting of Vamp3, MHC class I, β1-integrin and β1-integrinreceptor are present in the sample.
 12. A method of identifying invasivemicrovesicles in a sample from a patient suffering from a tumor, themethod comprising the steps of: obtaining a biological sample from thepatient; centrifuging the biological sample at from 5,000 g to 15,000 gto obtain a population of microvesicles, and assaying the population ofmicrovesicles for presence of the protein ARF6, wherein the presence ofARF6 indicates that the sample comprises invasive microvesicles.
 13. Themethod of claim 12, additionally comprising assaying the population ofmicrovesicles for the presence of MT1-MMP.
 14. The method of claim 12,wherein the sample is centrifuged two or more times at from 5,000 g to15,000 g.
 15. The method of claim 12, additionally comprisingcentrifuging the sample at from about 1,200 g to about 3,000 g prior tocentrifuging the sample at from 5,000 g to 15,000 g.
 16. The method ofclaim 12, wherein the population of microvesicles is assayed for thepresence of ARF6 by western blot.